1. Field of the Invention
The present invention relates to a non-naturally-occurring amino acid-incorporated protein expression method by which a non-naturally occurring amino acid is incorporated at a desired site in a protein, as well as DNA, an expression vector and animal cells used for expressing the aforementioned protein.
2. Description of the Related Art
Non-naturally-occurring amino acid-containing proteins (to also be referred to as alloproteins), in which an amino acid residue at a desired location in a protein is substituted with an amino acid other than the 20 types of amino acids normally involved in protein synthesis (to be referred to as non-naturally-occurring amino acids), offer an effective means of analyzing the function and structure of proteins.
Aminoacyl t-RNA synthetases (to be referred to as aaRS) are enzymes that specifically bind amino acids and tRNA, and excluding a few exceptions, there are 20 types of such enzymes corresponding to each of the 20 types of amino acids that exist in nature for each biological species. Since these aaRS are basically present for each amino acid within cells, the type of amino acid assigned to the genetic code is determined. For example, TyrRS, which is a kind of aaRS (to simply be referred to as TyrRS), distinguishes tyrosine tRNA (to be referred to as tRNATyr) from the tRNA of other amino acids, and binds only tyrosine to tRNATyr without binding other amino acids.
Known methods for producing alloproteins in the prior art consist of a method that produces alloproteins in Escherichia coli (Koide, et al., Proceedings of the National Academy of Science USA, Vol. 85, 1988, pp. 6237-6241 (Document 1)), and a method that produces alloproteins in a cell-free translation system (Noren, et al., Science, Vol. 244, 1989, pp. 182-188 (Document 2)).
In order to prepare proteins containing 21 types of amino acids, including non-naturally-occurring amino acids, in large yield, it is necessary to construct an artificial genetic code system by which tRNAs are aminoacylated with non-naturally-occurring amino acids by specific aaRSs in a system in which a translation reaction takes place.
It is necessary to find aaRS-tRNA pairs that satisfy the following conditions in order to construct such an artificial genetic code system:
(1) the aaRS must be an aaRS mutant that reacts specifically with a desired non-naturally occurring amino acid and not with any of the ordinary 20 types of amino acids; and,
(2) the tRNA must be assigned to a codon to which none of the ordinary 20 types of amino acids are assigned (such as a nonsense codon or 4-base codon), must be recognized only by the aforementioned aaRS mutant specific for a non-naturally-occurring amino acid, and must not be recognized by any aaRS of the host (orthogonal tRNA).
A tRNA molecule that binds a non-naturally-occurring amino acid and transport it to a nonsense codon on messenger RNA (suppressor tRNA molecule), and an enzyme that binds a non-naturally-occurring amino acid to this suppressor tRNA molecule (aaRs), can be used to create a genetic code system that satisfies these conditions. This mechanism is described in Wang, et al., Science, Vol. 292, 2001, pp. 498-500 (Document 3) and Journal of the American Chemical Society, Vol. 124, 2002, pp. 1836-1937 (Document 4).
Specific artificial genetic code systems based on this mechanism have been established in E. coli as well as cell-free protein synthesis systems using wheat germ extract.
Namely, a system for producing protein containing a non-naturally-occurring amino acid at an arbitrary specified site in E. coli has been reported that consists of O-methyltyrosine being specifically inserted corresponding to an amber codon by expressing a TyrRS mutant originating in Methanococcus jannaschii that has been altered so as to specifically aminoacylate O-methyltyrosine, and an amber suppressor tRNA in which tyrosine tRNA from the same microorganism has been altered (Document 3).
In addition, aaRS has been developed for producing protein containing a non-naturally-occurring amino acid at an arbitrary site in wheat germ extract (Kiga, et al., Proceedings of the National Academy of Science USA, Vol. 99, Jul. 23, 2002, pp. 9715-9723 (Document 5)).
In this manner, although alloprotein production systems have been developed in E. coli and cell-free protein synthesis systems, these cannot be used directly in animal cells. Namely, these molecules developed for use in E. coli cannot be used in animal cells due the properties of the molecules themselves. In order to synthesize protein containing a non-naturally-occurring amino acid at an arbitrary specified site in animal cells, suitable suppressor tRNA and aaRS specific for both this suppressor tRNA and the non-naturally-occurring amino acid must be developed, and the expression of those molecules must be realized in animal cells.
In addition, the aforementioned aaRS used in a cell-free protein synthesis system in wheat germ extract is predicted to be able to be used in animal cells in consideration of its substrate specificity. However, in order to use this aaRS, it is necessary to develop suppressor tRNA to be combined therewith along with the expression system for this tRNA.